BB0259 Encompasses a Peptidoglycan Lytic Enzyme Function for Proper Assembly of Periplasmic Flagella in Borrelia burgdorferi

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector

The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a family of modularly organized T6SS peptidoglycan-degrading effectors with an unprecedented impact in antagonistic bacterial interactions.

Molecular basis of dimerization of lytic transglycosylase revealed by the crystal structure of MltA from Acinetobacter baumannii

Peptidoglycan digestion by murein-degrading enzymes is a critical process in bacterial cell growth and/or cell division. The membrane-bound lytic murein transglycosylase A (MltA) is a murein-degrading enzyme; it catalyzes the cleavage of the β-1,4-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycans. Although substrate recognition and cleavage by MltA have been examined by previous structural and mutagenesis studies, the overall mechanism of MltA in conjunction with other functionally related molecules on the outer membrane of bacterial cells for peptidoglycan degradation has remained elusive. In this study, the crystal structure of MltA from the virulent human pathogen Acinetobacter baumannii is characterized and presented. The study indicated that MltA from A. baumannii forms homodimers via an extra domain which is specific to this species. Furthermore, the working mechanism of MltA with various functionally related proteins on the bacterial outer membrane was modeled based on the structural and biochemical analysis.

Structural changes in bacteriophage T7 upon receptor-induced genome ejection

Many tailed bacteriophages assemble ejection proteins and a portal–tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal–tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near–atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

Modulation of the enzymatic activity of the flagellar lytic transglycosylase SltF by rod components, and the scaffolding protein FlgJ in Rhodobacter sphaeroides .

Macromolecular cell-envelope-spanning structures such as the bacterial flagellum must traverse the cell wall. Lytic transglycosylases enzymes are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. In the periplasmic space, the assembly of the flagellar rod requires the scaffold protein FlgJ, which includes a muramidase domain in the canonical models Salmonella enterica and Escherichia coli . In contrast, in Rhodobacter sphaeroides , FlgJ and the dedicated flagellar lytic transglycosylase SltF are separate entities that interact in the periplasm. In this study we show that sltF is expressed along with the genes encoding the early components of the flagellar hierarchy that include the hook-basal body proteins, making SltF available during the rod assembly. Protein-protein interaction experiments demonstrated that SltF interacts with the rod proteins FliE, FlgB, FlgC, FlgF and FlgG through its C-terminal region. A deletion analysis that divides the C-terminus in two halves revealed that the interacting regions for most of the rod proteins are not redundant. Our results also show that the presence of the rod proteins FliE, FlgB, FlgC, and FlgF displace the previously reported SltF-FlgJ interaction. In addition, we observed modulation of the transglycosylase activity of SltF mediated by FlgB and FlgJ that could be relevant to coordinate rod assembly with cell wall remodeling. In summary, different mechanisms regulate the flagellar lytic transglycosylase, SltF ensuring a timely transcription, a proper localization and a controlled enzymatic activity. Importance Several mechanisms participate in the assembly of cell-envelope-spanning macromolecular structures. The sequential expression of substrates to be exported, selective export, and a specific order of incorporation are some of the mechanisms that stand out to drive an efficient assembly process. In this work we analyze how the structural rod proteins, the scaffold protein FlgJ and the flagellar lytic enzyme SltF, interact in an orderly fashion to assemble the flagellar rod into the periplasmic space. A complex arrangement of transient interactions directs a dedicated flagellar muramidase towards the flagellar rod. All these interactions bring this protein to the proximity of the peptidoglycan wall while also modulating its enzymatic activity. This study suggests how a dynamic network of interactions participates in controlling SltF, a prominent component for flagellar formation.

The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia coli

Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.

Protein Family Content Uncovers Lineage Relationships and Bacterial Pathway Maintenance Mechanisms in DPANN Archaea

DPANN are small-celled archaea that are generally predicted to be symbionts, and in some cases are known episymbionts of other archaea. As the monophyly of the DPANN remains uncertain, we hypothesized that proteome content could reveal relationships among DPANN lineages, constrain genetic overlap with bacteria, and illustrate how organisms with hybrid bacterial and archaeal protein sets might function. We tested this hypothesis using protein family content that was defined in part using 3,197 genomes including 569 newly reconstructed genomes. Protein family content clearly separates the final set of 390 DPANN genomes from other archaea, paralleling the separation of Candidate Phyla Radiation (CPR) bacteria from all other bacteria. This separation is partly driven by hypothetical proteins, some of which may be symbiosis-related. Pacearchaeota with the most limited predicted metabolic capacities have Form II/III and III-like Rubisco, suggesting metabolisms based on scavenged nucleotides. Intriguingly, the Pacearchaeota and Woesearchaeota with the smallest genomes also tend to encode large extracellular murein-like lytic transglycosylase domain proteins that may bind and degrade components of bacterial cell walls, indicating that some might be episymbionts of bacteria. The pathway for biosynthesis of bacterial isoprenoids is widespread in Woesearchaeota genomes and is encoded in proximity to genes involved in bacterial fatty acids synthesis. Surprisingly, in some DPANN genomes we identified a pathway for synthesis of queuosine, an unusual nucleotide in tRNAs of bacteria. Other bacterial systems are predicted to be involved in protein refolding. For example, many DPANN have the complete bacterial DnaK-DnaJ-GrpE system and many Woesearchaeota and Pacearchaeota possess bacterial group I chaperones. Thus, many DPANN appear to have mechanisms to ensure efficient protein folding of both archaeal and laterally acquired bacterial proteins.